We have devised a new substrate patterning method that overcomes the limitations associated with random cell binding on pre-patterned substrates. Our approach provides the ability to create patterns that restrict, contain, allow or direct growth after cells have been plated. The ability to pattern substrates after plating allows selection of specific subsets of cells (i.e., transfected or knockout cells) and modification of patterns in response to changes in outgrowth. When combined with pre-patterned substrates comprising more than one material, polarization of neurons can be induced and interactions oriented. Using this approach we are able to create neuronal circuits with varying complexity and known connectivity. Our patterning technique is similar to maskless photolithography and uses a high-energy pulsed IR laser to directly unbind substrate materials. The areas where protein is removed become non-permissive for neuronal growth. We will use the combination of substrate patterning, microfluidics and electrophysiology to create and test functional neuronal circuits of hippocampal neurons. Once we have verified function and optimized the approach, we will focus on addressing specific biological questions. This will include studying the influence and regulation of neural activity on dendritic spine emergence, development and dynamics. We will also study the influence of an applied exogenous factor on spine formation and maturation, as well as alterations in spine dynamics in the absence of myosin IIB using cells from myosin IIB knockout mice. Finally, we will study the role of Rab5 in presynaptic function of hippocampal neurons connected in simple circuits. PUBLIC HEALTH RELEVANCE We have developed a new method of patterning substrates in the presence of living cells that allows creation of neuronal circuits with known connectivity in cell culture. This will advance our understanding of communication between nerve cells and facilitate modeling of brain circuits. This is important for understanding normal brain function and the potential for recovery from brain or spinal cord injury.